Spatial navigation in rodents is a widely used paradigm for studies of cognition, learning, and memory in the mammalian brain. The medial entorhinal cortex (MEC) has been recently shown to possess a representation of position in the form of spatial activity patterns of neurons in layer II, termed grid cells. Grid cells exhibit spatial firing patterns in the form of tessellated, equilateral triangles that cover the environment. Macroscopic patterning in the MEC has been observed where, across the dorsoventral extent, these triangles change in size. Locally, neighboring grid cells exhibit spatial firing patterns related by a phase shift, while patterns from grid cells a further distance away are related by both a phase shift and a change in orientation. How this representation of position is produced in the MEC is a fundamental and unanswered question. This proposal addresses three questions: does grid cell patterning arise from path integration of idiothetic cues (self-motion), how do grid cells perform path integration, and is there a functional microorganization of grid cells in layer II of MEC. To investigate the cellular and network mechanisms involved in navigation, the Tank lab has recently developed new technology for electrophysiological recordings and calcium imaging to be performed in the brain of awake mice during navigation in a virtual environment. This technology consists of a spherical treadmill that allows for motion in two dimensions, a virtual reality environment controlled by the motion of the animal on the spherical treadmill, and interchangeable equipment allowing for either electrophysiological recordings or optical imaging. This experimental setup has three advantages over conventional methods for studying navigation: 1.) The animal is head-fixed but allowed to navigate within a virtual environment, providing stability for whole cell recordings as well as calcium imaging. 2.) Only visual cues from the virtual environment;therefore, complete removal of allocentric (world) cues from a virtual environment is possible. 3.) The virtual environment can be dynamically altered during navigation tasks. This proposal takes advantage of these new technologies in order to understand how the MEC forms a map of the environment. In aim 1, tetrode recordings will be performed simultaneous with navigation in a virtual environment to establish the spatial firing properties of grid cells in this setting and to test whether grid cells are formed by an integration of self-motion cues such as heading and velocity. In aim 2, intracellular recordings will be performed during navigation in a virtual environment to investigate the intracellular dynamics of grid cells, such as the interaction between intracellular theta oscillations, spiking of the cell and running speed of the animal. In aim 3, calcium imaging will be performed to determine if there is a systematic spatial organization of cells in layer II defined by their phase and orientation. These new technologies will allow us to probe questions about brain function in a unique way and will open the doors to new questions as the ability to dynamically perturb the animal's environment based upon either its behavior or brain activity becomes feasible. PUBLIC HEALTH RELEVANCE: The medial entorhinal cortex is hypothesized to be the region of the mammalian brain responsible for the formation of a metric representation of the environment. Many diseases are associated with functional disruption of the medial entorhinal cortex, such as Alzheimer's disease, schizophrenia, and temporal lobe epilepsy. Understanding how the microcircuits in the MEC operate will provide a template for assaying disruptions to this patterning.